Mediator stabilized reagent compositions and methods for their use in electrochemical analyte detection assays

ABSTRACT

Mediator stabilized reagent compositions and methods for their use in electrochemical analyte determination assays are provided. The subject reagent compositions include an enzyme, a redox mediator and a mediator-stabilizing buffer. Optionally, the reagent compositions may further include one or more of a wetting agent, detergent, enzyme cofactor and combinations thereof. Also provided are electrochemical test strips that include the subject reagent compositions, systems and kits that include the same as well as methods for using the same in analyte detection assays. The subject invention finds use in a variety of different applications, including glucose concentration determination applications.

FIELD OF THE INVENTION

The field of this invention is analyte determination, particularlyelectrochemical analyte determination and more particularly theelectrochemical determination of blood analytes.

BACKGROUND

Analyte detection in physiological fluids or samples, e.g., blood orblood derived products, is of ever increasing importance to today'ssociety. Analyte detection assays find use in a variety of applications,including clinical laboratory testing, home testing, etc., where theresults of such testing play a prominent role in the diagnosis andmanagement of a variety of disease conditions. Analytes of interestinclude glucose for diabetes management, cholesterol, and the like. Inresponse to this growing importance of analyte detection, a variety ofanalyte detection protocols and devices for both clinical and home usehave been developed.

One type of method that is employed for analyte detection is anelectrochemical method. In such methods, an aqueous liquid sample isplaced into a reaction zone in an electrochemical cell comprising atleast two electrodes, i.e., a reference and working electrode, where theelectrodes have an impedance which renders them suitable foramperometric measurement. The component to be analyzed is allowed toreact directly with an electrode, or directly or indirectly with a redoxreagent to form an oxidizable (or reducible) substance in an amountcorresponding to the concentration of the component to be analyzed,i.e., analyte. The quantity of the oxidizable (or reducible) substancepresent is then estimated electrochemically and related to the amount ofanalyte present in the initial sample.

In many such electrochemical approaches to analyte detection, an analyteoxidizing signal producing system comprising an enzyme component and amediator component is employed, where the enzyme component oxidizes theanalyte of interest and then transfers an electron to a mediator which,in turn, transfers the electron to an electrode in the electrochemicalcell, thereby generating an electrical signal from which the analyteconcentration can be determined.

In electrochemical test strips, the strips are typically manufactured atsome time prior to their use. Between their manufacture and use, thetest strips are stored. During this storage period, a proportion of themediator can transform to its reduced from. In such situations,inaccurate results may be obtained when the strip is finally employedbecause some of the mediator is already reduced.

As such, there is interest in the development of electrochemical reagentformulations in which the mediator is storage stabilized. The presentinvention satisfies this need.

Relevant Literature

U.S. Pat. Nos. 5,723,284; 5,834,224; 5,942,102; 5,972,199; 5,997,817;6,059,946; 6,083,710; 6,121,009; 6,134,461; 6,179,979; 6,193,973 and6,284,125; as well as other patent documents: WO 99/49307; WO 97/18465;WO 01/57510; WO 01/57238; WO 02/48707; WO 02/50609; EP 0 969 097A2;JP091403378A; and GB 2 304 628.

SUMMARY OF THE INVENTION

Mediator stabilized reagent compositions and methods for their use inelectrochemical analyte determination assays are provided. The subjectreagent compositions include an enzyme, a redox mediator and amediator-stabilizing buffer. Optionally, the reagent compositions mayfurther include one or more of a wetting agent, detergent, enzymecofactor and combinations thereof. Also provided are electrochemicaltest strips that include the subject reagent compositions, systems andkits that include the same as well as methods for using the same inanalyte detection assays. The subject invention finds use in a varietyof different applications, including glucose concentration determinationapplications.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides an exploded view of an electrochemical test stripaccording to the present invention.

FIG. 2 shows the same test strip in assembled form.

FIG. 3 provides the results of linearity test comprising citrate, malicand tartaric acid formulations according to the present invention.

FIGS. 4 and 5 provide results showing the hematocrit effect forink-jetted citrate and mellitic acid formulations according to thepresent invention.

FIGS. 6 and 7 provide results showing the hematocrit performance forcitric acid and citraconic acid reagent formulations according to thepresent invention.

FIG. 8 provides results of a linearity test for citraconic and maleicacid reagent formulations according to the present invention.

FIGS. 9 and 10 show stability data for electrochemical glucose sensorsmade with the citraconate and mellitate reagent formulations accordingto the present invention.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Mediator stabilized reagent compositions and methods for their use inelectrochemical analyte determination assays are provided. The subjectreagent compositions include an enzyme, a redox mediator and amediator-stabilizing buffer. Optionally, the reagent compositions mayfurther include one or more of a wetting agent, detergent, enzymecofactor and combinations thereof. Also provided are electrochemicaltest strips that include the subject reagent compositions, systems andkits that include the same as well as methods for using the same inanalyte detection assays. The subject invention finds use in a varietyof different applications, including glucose concentration determinationapplications.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing the cell lines, vectors,and methodologies, which are described in the publications, which mightbe used in connection with the presently described invention.

As summarized above, the subject invention provides mediator stabilizedelectrochemical reagent compositions and methods for their use inelectrochemical analyte detection applications. In further describingthe subject invention, the subject reagent formulations are describedfirst in greater detail, followed by a review of electrochemical reagenttest strips that include the subject formulations and methods of usingthe same to electrochemically detect the present of an analyte, e.g.,quantitatively, in a sample. Finally, a review of representative systemsand kits according to the subject invention is also provided.

Mediator Stabilized Reagent Compositions

As summarized above, the subject invention provides mediator stabilizedelectrochemical reagent compositions. The subject electrochemicalreagent compositions are compositions that find use in electrochemicalanalyte detection devices, e.g., test strips, and are typical redoxreagent compositions. By mediator stabilized reagent composition ismeant a composition in which the mediator component of the compositiondoes not convert to a reduced form for an extended period of time undertypical storage conditions. By typical storage conditions is meant at atemperature ranging from about −20 to about 55, usually from about 5 toabout 40° C. and humidity ranging from about 5 to about 90%, usuallyfrom about 10 to about 60%. The subject mediator stabilized reagentcompositions are stable for storage periods ranging from about greaterthan 18 months.

The subject mediator stabilized electrochemical reagent compositionsaccording to the subject invention include at least the followingcomponents: an enzyme, a redox mediator and a mediator-stabilizingbuffer. The subject redox reagent compositions may further include oneor more additional components, including wetting agents, detergents,enzyme cofactors, and the like. Each of these components is nowdescribed separately in greater detail.

Enzyme Component

The enzyme component of the subject reagent compositions is, in manyembodiments, an enzyme or plurality of enzymes that work in concert tooxidize the analyte of interest. In other words, the enzyme member maybe made up of a single analyte oxidizing enzyme or a collection of twoor more enzymes that work in concert to oxidize the analyte of interest,allowing generation of the electrochemical signal detected. Enzymes ofinterest include oxidases, dehydrogenases, lipases, kinases,diaphorases, quinoproteins and the like.

The enzyme selected in the reaction depends on the particular analytefor which the electrochemical test strip comprising the enzyme isdesigned to detect. Representative enzymes include: glucose oxidase,glucose dehydrogenase, cholesterol esterase, cholesterol oxidase,lipoprotein lipase, glycerol kinase, glycerol-3-phosphate oxidase,lactate oxidase, lactate dehydrogenase, pyruvate oxidase, alcoholoxidase, bilirubin oxidase, uricase, and the like.

Redox Mediator

Another component of the reagent composition is a redox mediator, whichmay comprise one or more mediator agents. The mediator acts anintermediary that facilitates the transfer of electrons from the enzyme(which has taken one or more electrons from the analyte during analyteoxidation) to the electrode. A variety of different mediator agentsknown in the art may be used, including ferricyanide, phenazineethosulphate, phenazine methosulfate, phenylenediamine,N,N,N′,N′-tetramethyl phenylenediamine, 1-methoxy-phenazinemethosulfate, 2,5-dimethyl-1,4-benzoquinone,2,6-dimethyl-1,4-benzoquinone, 2,5-dichloro-1,4-benzoquinone, ferrocenederivatives, osmium bipyridyl complexes, ruthenium complexes and thelike. In many embodiments, the redox mediator is ferricyanide.

Mediator Stabilizing Buffer Component

Another component of the reagent composition is a mediator stabilizingbuffering component. The subject mediator stabilizing bufferingcomponents may be made up of one or more, e.g., two, three, four ormore, distinct buffering agents, where the buffering componentstabilizes the mediator during storage of the composition in dry formsuch that little if any of the mediator is reduced prior to use, e.g.,during storage. A buffer is considered to stabilize a mediator if, inthe presence of the buffer, little if any of the mediator converts to areduced form over a given storage period, as described above. Suitablebuffers are buffers that do not cause the background signal in anelectrochemical test to increase over time, as determined using theassays described in the Experimental Section, below. The backgroundsignal is the signal obtained when analyte free sample is introduced tothe electrochemical testing system.

The buffering component, when present in a fluid reaction mixtureprepared by combining the reagent composition with the sample to beassayed, is a component that maintains the pH of the reaction mixture atan acceptable range, where in many embodiments the buffering componentmaintains the pH range of the reaction mixture at a value ranging fromabout 4.0 to 8.0, for example from about 5.0 to about 7.5, such as fromabout 5.5 to about 7.0.

The one or more buffering agents of the buffering component have a pKathat provides for the above recited pH range in reaction mixturesprepared with the subject reagent formulations. Suitable bufferingagents typically have pKa values of about 4 to about 8, for example fromabout 4.5 to about 7.5 including from about 5.0 to 7.0, including fromabout 5.5 to 7.0. Certain types of buffering agents may have more thanone pKa value, and such types of agents may be employed, so long as atleast one of their pKa values falls within the above-described ranges.

The buffering agents employed in the subject reagent compositions shouldhave little, if any, binding affinity for divalent metal cations, e.g.,Ca²⁺, Mg²⁺, etc., such that they have a low propensity to producepolydentate binding complexes with divalent metal cations. A givenbuffering agent is considered to have a low binding affinity fordivalent metal cations if its binding affinity for divalent metalcations is less than the binding affinity of any enzyme/cofactor complexin the reaction mixture for the same divalent metal cations, where thebinding affinity of suitable buffering agents is typically at leastabout 2-fold, usually at least about 5-fold and more usually at leastabout 10-fold, e.g., 25-fold, 50-fold, etc., less than the bindingaffinity of any enzyme/cofactor complex in the reaction mixture for thesame divalent metal cation. The stability constant for the complexationof suitable buffering agents to divalent metal cations, particularlyCa²⁺, as measured using the assay described in Annali di Chimica, volume73, (1983), p. 619 typically does not exceed about 1500, usually doesnot exceed about 100 and more usually does not exceed about 5 mol⁻¹ dm³.

In certain embodiments, the buffering agents are small organicmolecules. By small is meant that the molecular weight of the subjectagents does not exceed about 5,000 daltons, and typically does notexceed about 2,500 daltons and more typically does not exceed about1,000 daltons, where in many embodiments the molecular weight of thebuffering agents ranges from about 50 to 750 daltons, e.g., from about75 to 500 daltons.

In one embodiment, the buffer agents are polycarboxylic acids. Bypolycarboxylic acids is meant that the buffering agents include two ormore carboxylic acid functional moieties, where the number of differentcarboxylic acid functional moieties may range from about 2 to about 10,e.g., from about 2 to about 8, including from about 2 to about 6. Thecarboxylic acid groups or functional moieties of the subject bufferingagents may be attached to a number of different structures, includingaliphatic, alicyclic, aromatic and heterocyclic structures. The presenceof more than one carboxylic acid group can have the beneficial effect ofproviding at least one pKa value for the buffer in the desired range.

In many embodiments, the two or more carboxylic groups of the subjectpolycarboxylic buffering agents are configured such that they stericallyhinder the polydentate binding of divalent metal ions, such as Ca²⁺ andthe like. For example, buffering agents having two or more carboxylicacid groups positioned in a cis-position on a stable backbone that doesnot permit movement of the cis-groups relative to each other, e.g., oneon ethylene backbone, etc., are of interest, where the stable backbonemay be part of a larger structure, e.g., an aromatic ring, etc.

In certain embodiments the buffering agents are described by thefollowing formula:

where R₁ and R₂ are independent H or organic moieties of one or morecarbon atoms, which may be linear or branched and substituted with oneor more heteroatoms, where R₁ and R₂ may be taken together to form aring structure, e.g., an aromatic ring structure, and the like.

Specific polycarboxylic acids of interest include, but are not limitedto: mellitic acid, citraconic acid, maleic acid, and the like, etc.

With respect to the above buffering agents, the buffering agents may bepresent in the subject reagent formulations as their free acids or saItsthereof, or both.

A feature of the subject buffering component is that that the agent(s)that makes up the buffering component is present in an amount sufficientto provide for the mediator stabilizing capacity, as described above. Influid compositions of the subject reagent formulations, theconcentration of the buffering component typically ranges from about 0.1to about 1000 mM, e.g., from about 0.5 to 500 mM. In certainembodiments, the buffer component is present at a low concentration,e.g., from about 0.5 to about 250 mM, usually from about 0.5 to about100 mM. In certain embodiments, the buffer component is present at ahigher concentration, e.g., from about 50 to about 500 mM. Where thereagent composition is a dry reagent formulation, e.g., as may bepresent in an electrochemical test strip as described in greater detailbelow, the amount of buffering component present in the dry compositiontypically ranges from about 0.01 to about 40.00, usually from about 1 toabout 10% wt/wt.

Optional Components

As indicated above, the reagent composition may further include one ormore of the following additional components: a wetting agent, detergent,coenzyme, enzyme cofactor, stabilizer, viscosity modifier orcombinations thereof.

Wetting Agent and Detergents

A wetting agent may be added, in some embodiments in combination with adetergent, to the reagent composition to facilitate uniform coating ofthe reagent composition onto an electrochemical test strip. A pluralityof one or more of the combination of agents may also be used. The agentsused may improve dissolution of the assay reagents as well as enhancethe wicking properties of a capillary fill strip. The agents includethose known in the art, for example, polymers, anti-foaming agents, andsurfactants. Representative types of surfactants/detergents of interestinclude, but are not limited to: Tritons, Macols, Tetronics, Silwets,Zonyls, and Pluronics. Suitable agents include Pluronic materials whichare block co-polymers of polyethylene oxide and polypropylene oxide.Examples of Pluronic materials include Pluronic P103 which has goodwetting properties and Pluronic F87 Prill which has good detergentproperties. Both Pluronic P163 and F87 Prill also have a cloud pointtemperature greater than 80° C. which is desirable since this propertyavoids a phase change in the composition during the drying process.

Enzyme Coenzymes

Coenzymes which activate the enzyme component of the subjectformulations may also be added to the reagent composition, wheredesired. An example of a coenzyme of interest is pyrroloquinolinequinone (PQQ). Other cofactors of interest include, but are not limitedto: nicotinamide adenine dinucleotide (NAD) flavin adenine dinucleotide(FAD), cytochrome, and the like, depending on the type of enzyme appliedto the test reagent.

Enzyme Cofactors

In certain embodiments, the subject compositions further include one ormore enzyme cofactors. Enzyme cofactors of interest include divalentmetal cations, e.g., Ca²⁺ Mg²⁺, etc.

Stabilizers

Stabilizers may also be added to the reagent composition to helpstabilize the enzyme and prevent denaturation of the protein. Thestabilizer may also help stabilize the redox state of the mediator, inparticular, the oxidized redox mediator. Examples of stabilizing agentsinclude, but are not limited to: carbohydrates (e.g., sucrose,trehalose, mannitol, and lactose), amino acids, proteins (such as BSAand albumin) and organic compounds such as EDTA and the like.

Viscosity Modifiers

Viscosity modifiers may also be added to the reagent to modify theliquid reagent rheology. Examples of such agents include poly(acrylicacid), poly(vinyl alcohol), dextran, BSA and the like.

Additional Features

The reagent composition may be present in a dry or liquid form. Theamounts of the various components as described above may vary, and thefollowing specifically provided amounts are provided for illustrationpurposes only.

In liquid formulations of the subject reagent compositions, the enzymecomponent is typically present in a concentration ranging from about 35to about 450, usually from about 130 to about 270 μM. The mediator istypically present in an amount ranging from about 250 to about 1000,usually from about 500 to about 1000 mM. The stabilizing buffercomponent is present in ranges as provided above. The concentration ofany coenzymes typically ranges from about 60 to about 670 μM, usuallyfrom about 200 to about 430 μM. The concentration of any cofactorstypically ranges from about 0.5 to about 5 mM.

In dry formulations, the amount of the enzyme component typically rangesfrom about 1.5 to about 15, usually from about 5 to about 10% dry wt/wt.The mediator is typically present in an amount ranging from about 60 toabout 85, usually from about 75 to about 85% dry wt/wt. The stabilizingbuffer component is present in ranges as provided above. The amount ofany coenzymes typically ranges from about 0.01 to about 0.1, usuallyfrom about 0.03 to about 0.06% dry wt/wt. The amount of any cofactorstypically ranges from about 0.02 to about 0.2% dry wt/wt.

Representative Specific Formulation of Interest

In a representative specific formulation of interest the formulationincludes glucose dehydrogenase as the enzyme and PQQ as a coenzyme. Alsopresent is an enzyme cofactor Ca²⁺. In these formulations, PQQ bindswith GDH and Ca²⁺ to form the activated enzyme, or holo-enzyme. It isknown that two PQQ molecules per GDH dimer molecule are required tofully activate the enzyme. In some embodiments, the mole ratio of PQQ toGDH in the reagent compositions is between about 2 to about 4. Infurther embodiments, the mole ratio of PQQ to GDH is between about 2.2to about 2.5. The activity of the GDH-PQQ holoenzyme in theserepresentative formulations typically ranges from about 10 to 1000kU/ml, usually at least about 50 to 300 kU/ml. The enzyme activity isdetermined using a spectrophotometric assay performed at 30° C.Typically, the 3 parts enzyme solution is mixed to 100 parts substratesolution. Next, the absorbance is monitored at 600 nm over a 5-secondperiod. The substrate solution comprises 112 mM glucose, 2 mM phenazineethosulfate, 60 nM 2,6-dichloroindophenol, and 50 mM piperazine, N,N′bis-(2-ethanesulfonic acid) (PIPES) at pH 6.8. The enzyme solutioncomprises about 0.25 to 0.50 mg/mL GDH, 1.25 μM PQQ, 1.25 mM CaCl2, 0.1%(w/v) bovine serum albumin, and 50 mM PIPES at pH 6.8. The amount ofCa²⁺, e.g., as provided by CaCl₂, typically ranges from about 0 to 10mM. The mediator in this specific formulation of interest isferricyanide, which is typically present in amounts ranging from about0.1 to 10 M, usually from about 0.3 to about 1.0 M. When present, thewetting agent/detergent component is present in an amount ranging fromabout 0.01 to 1.0% wt/wt. Sucrose, when present, is present in an amountranging from about 10 to about 1000 mM. The buffering agents of specificinterest are citraconic acid and or? mellitic acid, and these arepresent in the ranges provided above.

Electrochemical Cells

As summarized above, also provided by the subject invention areelectrochemical cells that include the subject reagent compositions. Avariety of different types of electrochemical cell configurations areknown, including those described in U.S. Pat. Nos. 5,723,284; 5,834,224;5,942,102; 5,972,199, 5,997,817; 6,083,710; 6,121,009; 6,134,461; and6,193,873; the disclosures of which are herein incorporated byreference; as well as other patent documents: WO 99/49307; WO 97/18465;WO 01/57510; WO 01/57238; WO 02/48707; WO 02/50609; EP 0 969 097A2 andGB 2 304 628; the priority documents of which, where they are U.S.applications, are herein incorporated by reference. Any of these orother electrochemical cells known to those of skill in the art may bemodified to incorporate the subject compositions.

In certain embodiments, the electrochemical cell is present in anelectrochemical test strip. A representation of an electrochemical teststrip according to the subject invention is provided in FIGS. 1 and 2.FIG. 1 provides an exploded view of an electrochemical test strip 10which is made up of working electrode 12 and reference electrode 14separated by spacer layer 16 which has a cutaway section 18 that definesthe reaction zone or area in the assembled strip. FIG. 2 shows the sametest strip in assembled form. Each of the various components are nowdescribed in greater detail below.

Electrodes

The subject electrochemical test strips comprising the reagentcompositions include a working electrode and a reference electrode.Generally, the working and reference electrodes are configured in theform of elongated rectangular strips. Typically, the length of theelectrodes ranges from about 1.9 to about 4.5 cm, usually from about 2to about 2.8 cm. The width of the electrodes ranges from about 0.38 toabout 0.76 cm, usually from about 0.51 to about 0.67 cm. The referenceelectrodes typically have a thickness ranging from about 10 to 100 nmand usually from about 18 to about 22 nm. In certain embodiments, thelength of one of the electrodes is shorter than the length of the otherelectrode, wherein in certain embodiments it is about 0.32 cm shorter.

The working and reference electrodes are further characterized in thatat least the surface of the electrodes that faces the reaction area inthe strip is a conductive material, e.g., a metal or other conductivematerial, where representative materials of interest include, but arenot limited to: palladium, gold, platinum, silver, iridium, carbon,doped tin oxide, stainless steel and the like. In certain embodiments,the conductive material is gold or palladium. While in principle theentire electrode may be made of the conductive material, each of theelectrodes is generally made up of an inert support material on thesurface of which is present a thin layer of the conducting materialcomponent of the electrode. Any convenient inert backing material may beemployed in the subject electrodes, where typically the material is arigid material that is capable of providing structural support to theelectrode and, in turn, the electrochemical test strip as a whole.Suitable materials that may be employed as the backing substrate includeplastics, e.g. PET, PETG, polyimide, polycarbonate, polystyrene,silicon, ceramic, glass, and the like.

Spacer Layer

A feature of the subject electrochemical test strips is that the workingand reference electrodes as described above face each other and areseparated by only a short distance, such that the distance between theworking and reference electrode in the reaction zone or area of theelectrochemical test strip is extremely small. This minimal spacing ofthe working and reference electrodes in the subject test strips is aresult of the presence of a thin spacer layer positioned or sandwichedbetween the working and reference electrodes. The thickness of thisspacer layer generally ranges from about 1 to about 500 μm, usually fromabout 100 to about 200 μm. The spacer layer is cut so as to provide areaction zone or area with at least an inlet port into the reactionzone, and generally an outlet port out of the reaction zone as well. Arepresentative spacer layer configuration can be seen in FIGS. 1 and 2.While the spacer layer is shown in these figures as having a circularreaction area cut with side inlet and outlet vents or ports, otherconfigurations are possible, e.g. square, oval, triangular, rectangular,irregular shaped reaction areas, etc. The spacer layer may be fabricatedfrom any convenient material, where representative suitable materialsinclude PET, PETG, polyimide, polycarbonate and the like, where thesurfaces of the spacer layer may be treated so as to be adhesive withrespect to their respective electrodes and thereby maintain thestructure of the electrochemical test strip. Of particular interest isthe use of a die-cut double-sided adhesive strip as the spacer layer.

Reaction Zone

The subject electrochemical test strips include a reaction zone or areathat is defined by the working electrode, the reference electrode andthe spacer layer, where these elements are described above.Specifically, the working and reference electrodes define the top andbottom of the reaction area, while the spacer layer defines the wails ofthe reaction area. The volume of the reaction area is at least about 0.1μl, usually at least about 1 μl and more usually at least about 1.5 μl,where the volume may be as large as 10 μl or larger. As mentioned above,the reaction area generally includes at least an inlet port, and in manyembodiments also includes an outlet port. The cross-sectional area ofthe inlet and outlet ports may vary as long as it is sufficiently largeto provide an effective entrance or exit of fluid from the reactionarea, but generally ranges from about 9×10⁻⁵ to about 5×10⁻³ cm²,usually from about 5×10⁻⁴ to about 2.5×10⁻³ cm².

Present in the reaction zone is a reagent formulation according to thepresent invention, where the reagent formulation is typically present ina dry format.

Analyte Detection Methods

Also provided by the subject invention are methods of using the subjectreagent compositions to determine the concentration of an analyte in aphysiological sample. For convenience, the methods are described interms of the above representative test strips. However, the invention isnot limited thereto, as any method of detecting an analyte using thesubject reagent formulations in an electrochemical cell is encompassedwithin the invention.

The methods include applying the sample to an electrochemical test stripthat includes the reagent compositions of the subject invention,detecting an electrical signal generated by the test strip and relatingthe detected electrical signal to the concentration of the analyte inthe sample. A variety of different analytes may be detected using thesubject test strips, where representative analytes include glucose,cholesterol, lactate, alcohol, and the like. In many preferredembodiments, the subject methods are employed to determine the glucoseconcentration in a physiological sample. While in principle the subjectmethods may be used to determine the concentration of an analyte in avariety of different physiological samples, such as urine, tears,saliva, and the like, they are particularly suited for use indetermining the concentration of an analyte in blood or blood fractions,and more particularly in whole blood.

In practicing the subject methods, the first step is to introduce aquantity of the physiological sample into the reaction area of the teststrip, where the electrochemical test strip is described supra. Theamount of physiological sample, e.g. blood, that is introduced into thereaction area of the test strip may vary, but generally ranges fromabout 0.05 to about 10 ul, usually from about 0.5 to about 1.6 ul. Thesample may be introduced into the reaction area using any convenientprotocol, where the sample may be injected into the reaction area,allowed to wick into the reaction area, and the like, as may beconvenient.

Following application of the sample to the reaction zone, anelectrochemical measurement is made using the reference and workingelectrodes. The electrochemical measurement that is made may varydepending on the particular nature of the assay and the device withwhich the electrochemical test strip is employed, e.g. depending onwhether the assay is coulometric, amperometric or potentiometric.Generally, the electrochemical measure will measure charge(coulometric), current (amperometric) or potential (potentiometric),usually over a given period of time following sample introduction intothe reaction area. Methods for making the above describedelectrochemical measurement are further described in U.S. Pat. Nos.4,224,125; 4,545,382; and 5,266,179; as well as WO 97/18465; WO99/49307; the disclosures of which are herein incorporated by reference.

Following detection of the electrochemical signal generated in thereaction zone as described above, the amount of the analyte present inthe sample introduced into the reaction zone is then determined byrelating the electrochemical signal to the amount of analyte in thesample. In making this derivation, the measured electrochemical signalis typically compared to the signal generated from a series ofpreviously obtained controls or standard values, and determined fromthis comparison. In many embodiments, the electrochemical signalmeasurement steps and analyte concentration derivation steps, asdescribed above, are performed automatically by a device designed towork with the test strip to produce a value of analyte concentration ina sample applied to the test strip. A representative reading device forautomatically practicing these steps, such that user need only applysample to the reaction zone and then read the final analyteconcentration result from the device, is further described in U.S. Pat.No. 6,193,873; the disclosure of which is herein incorporated byreference.

The methods may be employed to determine the concentration of a varietyof different analytes, where representative analytes include glucose,cholesterol, lactate, alcohol, and the like. In many preferredembodiments, the subject methods are employed to determine the glucoseconcentration in a physiological sample. While in principle the subjectmethods may be used to determine the concentration of an analyte in avariety of different physiological samples, such as urine, tears,saliva, and the like, they are particularly suited for use indetermining the concentration of an analyte in blood or blood fractions,e.g., blood derived samples, and more particularly in whole blood.

Systems

Also provided by the subject invention are systems for use in thedetection of analytes, where the systems include a reagent compositionaccording to the subject invention, e.g., present in a test strip asdescribed above, and a device for use in electrochemically assaying asample using the subject reagent compositions.

The devices or meters of the subject systems are typicallyelectrochemical measuring devices. The subject meters typically include:(a) a means for applying an electric potential to an electrochemicalcell into which the sample has been introduced; (b) a means formeasuring cell current in the cell; and (c) a means for relating thecurrent to the concentration of analyte in the cell. Representativeelectrochemical meters or devices are described in U.S. Pat. Nos.5,723,284; 5,834,224; 5,942,102; 5,972,199; 5,997,817; 6,083,710;6,121,009; 6,134,461; and 6,193,873; the disclosures of which are hereinincorporated by reference; as well as other patent documents: WO99/49307; WO 97/18465; WO 01/57510; WO 01/57238; WO 02/48707; WO02/50609; EP 0 969 097A2 and GB 2 304 628.

Kits

Also provided by the subject invention are kits for use in practicingthe subject methods. The kits of the subject invention include thereagent compositions as described above, where the compositions areoften present in a test strip, as described above. The subject kits mayfurther include an obtainment element for obtaining a physiologicalsample. For example, where the physiological sample is blood, thesubject kits may further include an element for obtaining a bloodsample, such as a lance for sticking a finger, a lance actuation means,and the like. In addition, the subject kits may include an analytestandard, e.g. a control solution that contains a standardizedconcentration of glucose. In certain embodiments, the kits also includean automated instrument, as described above, for use with the reagentcompositions and test strips that include the same.

Finally, the kits may include instructions for using the subjectcompositions in the determination of an analyte concentration in aphysiological sample. The instructions may be printed on a substrate,such as paper or plastic, etc. As such, the instructions may be presentin the kits as a package insert, in the labeling of the container of thekit or components thereof (i.e., associated with the packaging orsub-packaging) etc. In other embodiments, the instructions are presentas an electronic storage data file present on a suitable computerreadable storage medium, e.g., CD-ROM, diskette, etc.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Glucose Linearity Results Using Citrate, Malic,and Tartarate

Citric, malic, and tartaric buffer formulations were separatelyformulated at 100 mM concentration and pH 5.5. All 3 formulations alsocontained equivalent amounts of 0.1% anti-foam (RNA Equilibrator), 1 mMCaCl₂, PQQ (2× mole ratio to GDH), 200 mM potassium ferricyanide, and 45mg/mL GDH. Each formulation was ink-jetted onto a Pd substrate. Thesensors were tested with blood containing glucose usingchronoamperometry by applying a potential of −0.3 V for 10 sec, and thenapplying a potential of +0.3 V for 5 sec. Testing with blood showed goodlinearity with glucose for all cases (FIG. 3). Background for citrateand malic buffers were comparable while that of tartaric buffer washigher. In addition, tartaric buffer formed an insoluble salt with Ca²⁺making it less desirable.

Example 2 Glucose Linearity and Hematocrit Results Using Citrate andMellitate

Citrate buffer was formulated at 400 mM and pH 5.5. Mellitate buffer wasformulated at 160 mM and pH 6.4. Both formulations also containedequivalent amounts of 0.1% anti-foam (RNA Equilibrator), 1 mM CaCl₂, PQQ(2× mole ratio to GDH), 200 mM potassium ferricyanide, and 32 mg/mL GDH.Blood testing was conducted using three hematocrit levels (20, 42, and70%) and 4 glucose levels (FIGS. 4 and 5). The glucose response was notlinear, but did increase with increasing glucose concentration.Hematocrit performance for citrate buffer was slightly better thanmellitate. The results are provided in FIGS. 4 and 5.

Example 3 Hematocrit and Stability Results Using Citrate and Citraconate

Citrate and citraconate buffers were separately formulated at 300 mMconcentration and pH 6.5. Both formulations also contained equivalentamounts of 0.1% anti-foam (RNA Equilibrator), 4 mM CaCl₂, PQQ (2× moleratio to GDH), 800 mM potassium ferricyanide, and 46 mg/mL GDH.Formulations were deposited on Pd by means of inkjetting. Hematocritperformance for both citrate and citraconate buffers were similar (FIGS.6 and 7). Accelerated aging at 56° C. for 14 days showed thatcitraconate had excellent background and performance stability ascompared to citrate (Table 1). The bias represented in Table 1 is givenas an absolute response difference at the 40 mg/dL glucoseconcentration, and as a percentage bias for glucose concentrationgreater than 100 mg/dL. TABLE 1 CITRACONIC CITRATE Bias to YSI Bias toYSI GLUCOSE Bias to YSI at Day 14 Bias to YSI at Day 14 LEVEL at Day 0@56 C. at Day 0 @56 C. 40 6.98 5.38 4.57 −7.06 240 6.52 3.69 1.44 −25.77540 0 1.45 0 −35.35

Example 4 Linearity and Stability Results Using Citraconate and Maleic

Citraconate and maleic buffers were separately formulated at 300 mMconcentration and pH 6.5. Both formulations also contained equivalentamounts of 0.066% Pluronic 25R2, 0.033% Pluronic L62, 4 mM CaCl₂, PQQ(2× mole ratio to GDH), 800 mM potassium ferricyanide, and 26 mg/mL GDH.Formulations were deposited on Pd by means of inkjetting. Initialperformance testing with nominal hematocrit showed good linearity (FIG.8). Stability data indicated stable background in both cases; however,performance for maleic buffer formulation degraded at the high glucoselevels—an indication of enzyme instability (Table 2). TABLE 2 CITRACONICMALEIC Bias to YSI Bias to YSI GLUCOSE Bias to YSI at Day 14 Bias to YSIat Day 14 LEVEL at Day 0 @56 C. at Day 0 @56 C. 40 6.17 4.52 0.03 −0.93240 7.29 3.93 −0.26 −10.93 540 −3.41 −0.08 −12.20 −25.37

Example 5 Hematocrit and Stability Results Using Citraconate andMellitic Buffers

Strips were made in a manner similar to Example 4 except that thereagent was deposited by a manual pipette and dried under a hot platewith a hot air stream. FIGS. 9 and 10 show the stability data forglucose sensors made with citraconate and mellitate buffers. A pH of 6.5was chosen with a buffer concentration of 105 and 40 mM for melliticacid and citraconic acid, respectively. The sensors were stored at 5° C.or 56° C. for 2 weeks and then tested with 18, 37 and 63% hematocritblood.

The above results and discussion demonstrate that the present inventionprovides electrochemical reagent compositions in which the mediator isstorage stabilized. Advantages of the subject invention include moreaccurate results, as well as obviation of the need to include lessdesirable stabilizing agents and/or perform a burn and read protocol. Assuch, the subject invention represents a significant contribution to theart.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference. The citation of any publication is for its disclosure priorto the filing date and should not be construed as an admission that thepresent invention is not entitled to antedate such publication by virtueof prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1-38. (canceled)
 39. A dry reagent composition comprising: an enzyme; aredox mediator; an enzyme cofactor; and a mediator-stabilizing buffercomprising a polycarboxylic acid having at least two carboxylic acidgroups, present in an amount in the range of 0.01% wt/wt to 40% wt/wtsufficient to stabilize said redox mediator during storage of the dryreagent composition, wherein said mediator-stabilizing buffer has a lowbinding affinity for divalent metal ions, said low binding affinity fordivalent metal ions being at least 2-fold less than a divalent metal ionbinding affinity of an enzyme/cofactor complex formed from the enzymeand the cofactor of the dry reagent mixture.
 40. The reagent compositionaccording to claim 39, wherein said polycarboxylic acid has from 2 to 10carboxylic acid groups.
 41. The dry reagent composition according toclaim 39, wherein said buffer has a pKa ranging from about 5 to about 8.42. The dry reagent composition according to claim 41, wherein saidbuffer has a pKa ranging from about 6 to about
 7. 43. The dry reagentcomposition according to claim 39, wherein said buffer is formulated tohave a pH in the range from about 4 to
 8. 44. The dry reagentcomposition according to claim 43, wherein said buffer is formulated tohave a pH in the range from about 6 to
 7. 45. The dry reagentcomposition according to claim 39, wherein at least two of saidcarboxylic groups are present in a cis formation.
 46. The dry reagentcomposition according to claim 45, wherein at least two of saidcarboxylic groups present in a cis formation are bonded to an ethylenemoiety.
 47. The dry reagent composition according to claim 46, whereinsaid polycarboxylic acid is mellitic acid and salts thereof.
 48. The dryreagent composition according to claim 39, wherein said enzyme is anoxidizing enzyme.
 49. The dry reagent composition according to claim 48,wherein said oxidizing enzyme is chosen from an oxidase and adehydrogenase.
 50. The dry reagent composition according to claim 48,wherein said enzyme is a dehydrogenase.
 51. The dry reagent compositionaccording to claim 48, wherein said oxidizing enzyme is a glucoseoxidizing enzyme.
 52. The dry reagent composition according to claim 39,wherein said mediator is ferricyanide.
 53. The dry reagent compositionaccording to claim 39, wherein said reagent composition furthercomprises a stabilizer.
 54. The dry reagent composition according toclaim 53, wherein said stabilizer is a carbohydrate.
 55. The dry reagentcomposition according to claim 54, wherein said carbohydrate is sucrose.56. An electrochemical cell comprising a dry reagent composition thatincludes: an enzyme; a redox mediator; an enzyme cofactor; and amediator-stabilizing buffer comprising a polycarboxylic acid having atleast two carboxylic acid groups, present in an amount in the range of0.01% wt/wt to 40% wt/wt sufficient to stabilize said redox mediatorduring storage of the dry reagent composition, wherein saidmediator-stabilizing buffer has a low binding affinity for divalentmetal ions, said low binding affinity for divalent metal ions being atleast 2-fold less than a divalent metal ion binding affinity of anenzyme/cofactor complex formed from the enzyme and the cofactor of thedry reagent mixture.
 57. The electrochemical cell according to claim 56,wherein said cell is present in an electrochemical test strip.
 58. Amethod for making an electrochemical test strip, the method comprising:making an electrochemical strip that includes: a dry reagent compositionwith: an enzyme; a redox mediator; an enzyme cofactor; and amediator-stabilizing buffer comprising a polycarboxylic acid having atleast two carboxylic acid groups, present in an amount in the range of0.01% wt/wt to 40% wt/wt sufficient to stabilize said redox mediatorduring storage of the dry reagent composition, wherein saidmediator-stabilizing buffer has a low binding affinity for divalentmetal ions, said low binding affinity for divalent metal ions being atleast 2-fold less than a divalent metal ion binding affinity of anenzyme/cofactor complex formed from the enzyme and the cofactor of thedry reagent mixture.
 59. A method for determining the concentration ofan analyte in a sample, said method comprising: (a) applying said sampleto an electrochemical cell comprising a dry reagent composition thatincludes: an enzyme; a redox mediator; an enzyme cofactor; and amediator-stabilizing buffer comprising a polycarboxylic acid having atleast two carboxylic acid groups, present in an amount in the range of0.01% wt/wt to 40% wt/wt sufficient to stabilize said redox mediatorduring storage of the dry reagent composition, wherein saidmediator-stabilizing buffer has a low binding affinity for divalentmetal ions, said low binding affinity for divalent metal ions being atleast 2-fold less than a divalent metal ion binding affinity of anenzyme/cofactor complex formed from the enzyme and the cofactor of thedry reagent mixture; (b) detecting an electrical signal produced by saidcell; and (c) relating said detected electrical signal to theconcentration of said analyte in said sample.
 60. A method according toclaim 59, wherein said analyte is glucose.
 61. A method according toclaim 59, wherein said physiological sample is blood.